Antibody for diagnosing neuropsychiatric diseases, in particular schizophrenia, depression and bipolar affective disorders

ABSTRACT

An antibody for diagnosis or treatment of neuropsychiatric diseases, in particular schizophrenia, depression or bipolar affective disorders, is characterized in that the antibody recognizes misfolded specific proteins that can be assigned to one of the diseases, and a method for diagnosis by means of antibodies that bind to neuropsychiatric disease-specific proteins.

This application is a national stage filing pursuant to 35 U.S.C. 371 ofPCT/EP05/01371, having an International filing date of Feb. 11, 2005 andclaiming the benefit of priority from the German patent application DE10 2004 007 462.3, filed Feb. 13, 2004, all of which are incorporated byreference. The present application claims the benefit of these priorapplications, including the right to priority.

BACKGROUND OF THE INVENTION

The present invention relates to antibodies for diagnosis and treatmentof neuropsychiatric diseases. The invention essentially relates toneuropsychiatric diseases, such as schizophrenia, depression or bipolaraffective disorders. The present application focuses essentially onschizophrenia. However, the statements thus made also pertain generallyto other neuropsychiatric diseases.

Schizophrenia is a neuropsychiatric disease likely to have heterogeneouscauses. From studies on twins it has meanwhile become accepted thatbiological causes are responsible for the development of schizophrenia.In terms of neuropathology, patients with schizophrenia arecharacterized by an expansion of the third cerebral ventricle which isconsidered to be an unspecific sign of a loss of brain structure.Schizophrenia, at least one sub-group of schizophrenia, namely thosewith pronounced so-called negative symptoms, can be classified amongstthe neurodegenerative diseases (J. Lieberman 1999, Biological Psychiatry46: 729f).

To date, no diagnosis of schizophrenia or other neuropsychiatricdiseases can be made on the basis of biological criteria. In order todetermine a diagnosis, suitably trained physicians (psychiatrists,neurologists) interview for certain main psychiatric symptoms; this canbe made more objective by means of so-called check lists. Unlikediseases in the area of internal medicine or neurology, the diseasecannot be diagnosed by means of unambiguous blood or liquor tests orimaging procedures. This leads to some uncertainty in determining thediagnosis.

From WO 0026675 a generic method for diagnosis of neuropsychiatricdiseases is known, in which the presence of polyglutamine-containingproteins in a tissue or body fluid sample of a patient is tested bymeans of an antibody that is directed against polyglutamine-containingprotein domains. Further, WO 0026675 specifies that the neuropsychiatricdiseases to be diagnosed with this method can be schizophrenia.

Polyglutamine-containing proteins are present in a plurality ofneurodegenerative diseases, e.g. Huntington's disease or spinocerebellarataxias. These diseases are characterized by increased occurrence ofrepeating glutamine residues in one or more proteins due to mutation,and are also referred to collectively as CAG repeat diseases since theDNA triplet, CAG, codes for glutamine.

In this context, for example, in Huntington's disease, a mutation in thehuman HD gene leads to an increase in the number of glutamine residuesat the N-terminus of the huntingtin protein. Because of its erroneousamino acid sequence, the polyglutamine-rich mutant huntingtin tends toaggregate with other polyglutamine-rich huntingtin molecules. In theprocess, agglomerates are formed in the cellular nucleus of neurons thatare associated with the fatal course of the disease. Similarsequence-related protein aggregations are thought to be responsible forthe generation of disease-specific symptoms of other polyglutaminediseases.

The method known from WO 0026675 uses a monoclonal antibody (1C2) thatis directed against polyglutamine-containing protein domains and istherefore not specific for schizophrenia. The method is only suitablefor detecting polyglutamine-containing proteins. However, there is somedoubt as to whether or not schizophrenia is even associated with theoccurrence of polyglutamine-containing proteins. These doubts shall beelaborated in more detail below.

On the presumption—postulated in WO 0026675, but neverthelessincorrect—that schizophrenia is associated with the presence ofpolyglutamine-containing proteins, the known method could, thus, help insupporting a suspicion of schizophrenia that is based on a psychiatricdiagnosis. However, diagnosis of schizophrenia through the use of thismethod alone is not feasible.

BRIEF SUMMARY OF THE INVENTION

It is the object of the present invention to provide antibodies fordiagnosis or treatment of neuropsychiatric diseases, in particular ofschizophrenia, depression or bipolar affective disorders. Further, it isan object of the present invention to provide a method that can be usedto reliably and unambiguously diagnose said neuropsychiatric diseases,in particular schizophrenia. And lastly, it is another object of thepresent invention to provide pharmaceutical compositions for treatmentof, for example, schizophrenia.

According to the invention, an antibody is provided that generallyrecognizes misfolded proteins that can be assigned to a neuropsychiatricdisease. Preferably, antibodies recognizing misfolded proteins that arespecific for schizophrenia, depression or bipolar affective disordersare provided.

For the purposes of the invention, the term “neuropsychiatric disease”shall cover in particular psychoses, including organic psychoses, i.e.diseases comprising classical features of psychosis as symptoms(delusion, hallucinations, impairment of thought, mood changes or otheraffective symptoms and cognitive disorders). It is particularlypreferred for this to concern schizophrenia, depression, and bipolardisorders.

In a further development, it is also conceivable that an antibodyaccording to the invention recognizes misfolded proteins that arespecific for multiple neuropsychiatric diseases, whereby the assignmentto a disease is possible by means of further properties of the protein,for example, its solubility or its molecular weight, or by means of theorigin of the protein, for example, from a certain region of the brain.

A preferred development provides the antibody according to the inventionto be obtained by immunization of suitable animals with purified brainfractions of patients afflicted by a neuropsychiatric disease, wherebysteps are provided in the purification that effect an enrichment ofmisfolded proteins.

In order to illustrate the antibody according to the invention in moredetail, it is necessary to first refer again to the polyglutaminediseases mentioned above. Since these are due to a mutation in a gene,as mentioned above, the predisposition for these diseases is usuallyhereditary. For this reason, they are collectively called “hereditaryneurodegenerative disorders”. However, it is a contentious issue whetheror not for example schizophrenia even is a polyglutamine disease, since,unlike Huntington's disease, no polyglutamine-containing protein hasbeen identified, thus far, that can be associated with the developmentof schizophrenia or is detected in schizophrenia patients at anomalousconcentrations.

Moreover, polyglutamine diseases are rare diseases. Compared toHuntington's disease, neurodegenerative diseases that are not associatedwith polyglutamine are significantly more common, e.g. Alzheimer'sdisease 150-fold, Parkinson's disease 30-fold, and schizophrenia100-fold more commonly (S. Prusiner 2001, New England Journal ofMedicine 344: 1516f).

Although it is accepted that there exists a genetic disposition forschizophrenia, the causes appear to be multifactorial. This is indicatedby the fact that even identical twins of a patient afflicted byschizophrenia have an only 50% risk of also being afflicted byschizophrenia (I. Gottesman and J. Shields 1982, Schizophrenia: theepigenetic puzzle, Cambride University Press, Cambridge; Kendler et al1993, The Roscommon Family Study. I. Methods, diagnosis of probands, andrisk of schizophrenia in relatives, Arch Gen Psychiatry 50 (7): 527).

The inventors then are the first to have found evidence indicating thatthe presence of misfolded proteins can serve as a diagnostic marker ofschizophrenia or other neuropsychiatric diseases, such as for example,depression or bipolar affective disorders. In the process, it waspostulated that misfolding is due to posttranslational causes and cannot, or to a small degree only, be associated with sequence-relatedanomalies such as CAG repeats. For example, an error during proteinbiosynthesis or protein processing in the endoplasmic reticulum possiblythat can be caused by defective processing enzymes, such as for example,translocon components or chaperones, can be responsible for misfolding.It is also conceivable that repair enzymes or proteases intended torepair and/or digest already misfolded proteins, are defective infunction. It is also conceivable that certain posttranslationalprocessing enzymes or proteases incorrectly modify an initiallycorrectly folded protein such that it is produced excessively withincorrect folding.

In this context, the defective functions of the processing enzymes,repair enzymes and/or proteases can be caused by mutations in therespective genes or their interaction partners on the protein levelwhich might explain a possible familial disposition for schizophrenia.In this context, possible candidates being discussed include the genesfor the proteins, DISC-1, dysbindin, neuregulin 1, COMT, RGS4,metabotropic glutamate receptor-3, DMO, and G72 (P. Harrison & M. Owen2003, Lancet 361:417f; P. Harrison and D. R. Weinberger 2005, MolecularPsychiatry 10:40-68).

According to the invention, the brain fractions from, for exampleschizophrenia, patients, are purified in a targeted fashion in order toisolate and enrich the posttranslationally misfolded proteins. In apreferred development, the purified fractions can be used to immunize,according to conventional technique, suitable animals (rabbits, mice,sheep, chickens) and, thus, obtain antibodies that bind specifically tothese proteins.

Instead of an immunization, it is feasible to use recombinant ligand orantibody libraries, that are expressed, for example, in phages, toidentify suitable anti-bodies or antibody fragments that bindspecifically to the purified misfolded proteins and can beaffinity-matured by mutagenesis under in vitro conditions after theiridentification.

A particularly preferred development provides the purification to be apurification step with ionic detergents. The purification of the brainfractions with ionic detergents serves to dissolve easily denaturedproteins in the sample to be purified. However, misfolded proteins witha tendency to aggregate are not denatured by ionic detergents, inparticular at temperatures between 0 and 10 degrees centigrade and,thus, do not remain in solution, i.e. pellet under the conditions of(ultra-)centrifugation and can, thus, be isolated in native form for theimmunization.

In order to exclude that the schizophrenia-specific proteins arepolyglutamine-containing proteins, the applicant subjected brainfractions that were isolated according to the invention fromschizophrenia patients and healthy test subjects to a Western blot usingthe labeled antibody, MW1. Similar to the antibody, 1C2, that was usedin WO 0026675, this antibody recognizes polyglutamine-rich epitopes inproteins and/or polyglutamine polymers. These experiments showed thatpolyglutamine-containing proteins were detected both in the brainfractions of schizophrenia patients and in those of healthy testsubjects. However, no differences in the polyglutamine content weredetected. The results are discussed below. The applicant concluded fromthese results that polyglutamine-containing proteins do not occur atelevated concentration in schizophrenia patients and thus cannot be usedas diagnostic markers for schizophrenia.

As illustrated above, the purification of the brain fractions with ionicdetergents serves to isolate misfolded proteins that are not denaturedby these detergents. Moreover, a preferred development provides thepurification step to be carried out at 0-10° C. since, at elevatedtemperatures, misfolded proteins can be denatured by ionic detergentsalso. It is particularly preferred to use a temperature of 0-5C in thispurification step.

A further preferred development of the invention provides the ionicdetergents that are used during the purification step to be used at aconcentration between 0.2 and 2%. This measure again serves to preventdenaturation of the misfolded proteins, since these, at elevatedconcentration, can be denatured by ionic detergents also. Preferably aconcentration is used in this purification step that ensures that thedetergent does not yet form micelles (“critical micellar concentration”,CMC).

Detergent molecules bind to proteins below the CMC, but form micellesabove the CMC. A concentration below the CMC is more beneficial for thepurification of misfolded proteins, since more detergent binds toundesired proteins and dissolves them, unlike misfolded proteins. TheCMC differs between detergents. In addition, it must be taken intoconsideration that the CMC is also dependent on the pH value andtemperature of the medium. Preferably, a concentration between 0.2 and1% is used in this purification step.

A further preferred development provides the ionic detergent used in thepurification step to be sarcosyl. The use of sarcosyl is preferredbecause it facilitates, on the one hand, the sufficient denaturation ofundesired, correctly folded proteins, and, on the other hand, preservesthe microaggregates of the misfolded proteins. Thisaggregation/solubility balance depends on the CMC of a detergent.

In contrast, the detergent, sodium dodecyl sulfate (SDS), acts toostrongly denaturing and is therefore less well suited for the statedpurpose.

It is particularly preferred to use sarcosyl at a concentration rangebetween 0.3 and 0.42%. Under normal conditions, sarcosyl reaches its CMCat 0.45%.

It is particularly preferred to use an ultracentrifugation step at least100,000×g in the purification step. Since the misfolded proteins areinsoluble under the conditions mentioned, they are found in the pelletafter ultracentrifugation, whereas other proteins remain in thesupernatant.

A further preferred development provides the purification to include apurification step with β-sheet-binding substances, such as Congo red,thioflavine or β-sheet-binding peptides. If applicable, these substancescan be immobilized in a chromatography column or similar. Since thesecondary structure of the misfolded, schizophrenia-specific proteinscomprises an increased fraction of β-sheet domains (“beta sheet”), theproteins thus sought can be specifically enriched by this means.

It is also preferred to provide a protease digestion step at atemperature between 0-10° C. in the purification. This step is alsosuitable for enriching misfolded schizophrenia-specific proteins sincethese possess increased resistance to proteases, such as proteinase K,at low temperatures due to their folding, whereas non-misfolded proteinsare digested by proteases at these temperatures.

A further preferred embodiment provides the antibody to be a monoclonalantibody. In order to obtain monoclonal antibodies, a suitable animal isimmunized first and then antibody-producing cells (e.g. B cells from thespleen) are removed from the immunized animal, fused with immortalizedmyeloma cells, and subjected to selection. The hybridoma cells thusobtained are then selected according to the specificity for themisfolded protein of the antibodies they produce. Instead of animmunization, it is feasible to use recombinant ligand or antibodylibraries, that are expressed, for example, in phages, to identifysuitable antibodies that bind specifically to the purified misfoldedproteins and can be affinity-matured by mutagenesis under in vitroconditions after their identification.

In particular, monoclonal antibodies termed 7B2 and 9C9 can be usedaccording to the invention. Hybridoma cells that can be used to producethe antibodies have been deposited in accordance with the Budapestagreement using the numbers, DSM ACC2713 and DSM ACC2714.

Monoclonal antibodies provide for higher specificity in theimmunochemical detection reaction and, thus, improve the accuracy of,for example, a detection method. In this context, it is particularlypreferred to provide the antibody to be a conformation-specificmonoclonal antibody, i.e. an antibody that recognizes an epitope of agiven protein only when it has a certain conformation, for example,exclusively when the epitope is present in a β-sheet conformation.

A particularly preferred development provides the antibody to be arecombinant antibody. In order to obtain an antibody of this type, DNAis isolated, e.g. from the spleen cells of immunized animals, and theparatope-coding cDNA fragments are cloned subsequently.

A further preferred development provides the antibody to be ablood-brain barrier-crossing antibody. The term, blood-brainbarrier-crossing, means that the antibodies can cross the blood-brainbarrier. In this context, various options are conceivable for renderingthe blood-brain barrier such that it can be crossed by antibodies, forexample the concomitant administration of suitable pharmaceuticals orhypertonic sugar solutions. On the other hand, the antibodies can aswell be rendered capable of crossing the blood-brain barrier bymolecular biological modifications, for example, by increasing theirhydrophobicity or lowering their molecular weight or masking theantibody through a signal sequence that promotes the targeted transportacross the blood-brain barrier.

Monoclonal antibodies possess very much higher specificity thanpolyclonal antibodies, but, like the former, also bear the risk ofrejection reactions in therapeutic use. A further preferred developmenttherefore provides the antibody to be a chimeric or a humanizedantibody. In chimeric antibodies, the constant domains, for example, ofmouse antibodies, are replaced by the corresponding constant domains ofhuman antibodies by molecular biological means. In humanized antibodies,in addition, the basic frameworks of the variable domain are replaced bycorresponding human sequences such that only the hypervariable regionsresponsible for antigen binding continue to be of murine origin.Antibodies, thus modified, cause only very weak and usually tolerablerejection reactions upon administration to the patient.

A further preferred development provides the antibody to be an antibodyfragment. This can concern, for example, monovalent F(ab) fragments,such as are obtained, for example, after papain digestion of IgGantibody molecules, or bivalent F(ab)₂ fragments such as are obtained,for example, after trypsin digestion. Antibody fragments are easier toclone into chimeric antibodies or to combine with humanized or humansequences and/or signal sequences. Moreover, antibody fragments cause aweaker rejection reaction.

A further development can provide the antibodies according to theinvention to be bound to a pharmaceutically active substance and/or,according to yet a further development, to an isotope or a radiolabeledmolecule. The latter antibodies can be used, for example, inradioimmunotherapy or nuclear medicine diagnostic work-up.

Further, a method is provided for diagnosis of, for example,schizophrenia or depression or bipolar affective disorders by means ofantibodies that bind to neuropsychiatric disease-specific proteins. Inthis context, the antibodies are contacted with a tissue or body fluidsample of a patient and antibody-protein complexes thus formed, if any,are detected. The potential presence of antibody-protein complexes isconsidered to be a positive finding of schizophrenia, depression orbipolar affective disorder in this context. The method is characterizedin that one of the antibodies claimed above is used.

In this context, it has become evident that the antibodies produced bythe applicant according to the invention bind specifically to proteinsthat are typical, for example, of schizophrenia. In order to provideevidence of this, the applicant carried out comparative immunochemicaltests using brain homogenates from healthy and schizophrenia-afflictedtest subjects. In this context, brain fractions from schizophreniapatients and healthy test subjects were subjected to a Western blotusing the antibodies obtained according to the invention. As a result,the applicant detected immunoreactive bands that were detected only inthe brain fractions from schizophrenia patients. Accordingly, theantibodies obtained according to the invention recognize proteins thatoccur only in schizophrenia patients. The results are discussed below.

A preferred development of the method provides the presence ofantibody-protein complexes to be detected by means of ELISA, Westernblotting or immuno-coupled fluorescence methods. However, any othersuitable method capable of detecting the binding of antibodies and/orother probe molecules to antigens, is conceivable as well.

Since, in the case of neuropsychiatric diseases, in particularschizophrenia, but also in depression or bipolar affective disease,misfolded proteins are presumably detectable in tissue or body fluidsamples of a patient long before the actual onset of disease, the methodaccording to the invention is also suitable for detection of a possiblypresent disposition for the respective neuropsychiatric disease, i.e.for schizophrenia, but also for depression or bipolar affective disease.Accordingly, a preferred development of the method according to theinvention provides the positive finding to be a diagnosed predispositionand/or a positive diagnosis for a specific neuropsychiatric disease,thus in particular for schizophrenia or a subgroup of schizophrenia,and/or depression or bipolar affective disorder.

A particularly preferred development of the method according to theinvention provides the body fluid sample to be tested to be liquor,urine, blood or serum. It can be presumed that disease-specificmisfolded proteins occur not only in the brain matrix, but also in bodyfluids of a patient suspected of schizophrenia, but also in patientssuspected of depression or bipolar affective disease. This applies inparticular to the liquor (cerebrospinal fluid) that is in constantcontact with the brain and of which samples can be obtained by tappingthe spinal canal as part of the clinical routine with no risk or pain.

However, the present invention is not limited to a method for diagnosisof schizophrenia, depression or bipolar affective disorder, but alsoextends to the use, for example, of the antibodies according to theinvention in the production of a pharmaceutical composition fortreatment of such diseases.

This concerns compositions that can be administered to a patient, inparticular, in a blood-brain barrier-crossing form. In particular, theantibodies claimed above are used in order for the antibodies, afterthey reach the brain, to bind to, for example, schizophrenia-specificmisfolded proteins and prevent, for example, their aggregation withother misfolded proteins.

A particularly preferred development provides the administeredantibodies to be coupled to pharmaceutically active substances.Substances of this type can, for example, be markers that mark theantibody-bound protein such that it can be digested by a protease orphagocytosed by a microglia cell. However, this can also concernsubstances that visualize the antibody-labeled proteins in imagingprocedures (NMR, CT). It is also conceivable to use antibodies coupledto radioactively labeled substances.

Another variant provides the production of compositions through the useof small-molecule, blood-brain barrier-crossing agents that recognizethe same sites as the antibodies claimed above that are directed againstmisfolded, schizophrenia-specific proteins. In this context, theantibodies claimed above serve as templates for definition of a surfacestructure on the misfolded proteins for which small-molecule agents arethen identified in chemical libraries by means of common techniques thatare collectively called “molecular design”, whereby said small-moleculeagents reproduce at least a similar surface structure on the misfoldedprotein as the antibody that is used. The antibodies can, for example,be molecules obtained by cloning, whose cDNA was identified from amolecular biological library of human antibodies or peptides.

Preferably, these small-molecule agents are organic molecules that bindspecifically to one of the epitopes of schizophrenia-specific proteinsthat are recognized by one of the antibodies claimed above. Such agentsare also called “small molecular drugs”. These can be natural substancesjust as well as molecules produced by synthesis. Small molecular drugsare advantageous as compared to anti-bodies or antibody fragments inthat they can be administered by the oral route, rarely elicitimmunological rejection reactions, and can cross the blood-brain barriermore easily due to their low molecular weight.

It is particularly preferred for these small molecule agents to comprisemultiple ligands that are connected to each other by spacers, wherebythe ligands each bind specifically to various, non-overlapping epitopesof schizophrenia-specific proteins that are recognized by the antibodiesclaimed above. Such agents are also called “composite molecules” and areadvantageous, in particular, as they possess much higher affinity forthe protein to be bound. Accordingly, the affinity of a molecule for theprotein to be bound multiplies with each new ligand added.

A final variant provides the production of pharmaceutical compositionsthrough the use of immunogenic substances that elicit an immune responsesuch that the immune system of the patient forms antibodies againstmisfolded, e.g. schizophrenia-specific, proteins.

These substances can, for example, be schizophrenia-specific proteins orproteins that are specific for other neuropsychiatric diseases such asdepression or bipolar affective disorder, such as can be isolated usingone of the purification methods claimed above or their equivalentsproduced by recombinant technique. In order to remove any pathogenicitythese proteins may have, it may under some circumstances be necessary tosubject them to a suitable treatment prior to administration. Inaddition, these substances can, for example, be fragments of theschizophrenia- (or, if applicable, depression- or bipolar affectivedisease-)specific proteins that are defined by means of the antibodiesclaimed above, whereby said fragments only contain the immunogenicregions, but are no longer pathogenic.

DETAILED DESCRIPTION OF THE INVENTION

The invention shall be illustrated in the following on the basis ofexamples:

A. Isolation of Misfolded Proteins

Solutions or Buffers Used (Sterile-Filtered):

VRL-buffer: 50 mM HEPES, pH 7.5, 250 mM sucrose, 5 mM MgCl₂, 100 mMKCH₃COO, 2 mM PMSF, protease inhibitor tablets, “Complete EDTA-free”(Roche 1873580)

High-sucrose buffer: 50 mM HEPES, pH 7.5, 1.6M sucrose, 100 mM KAc(KCH₃COO), 0.5% Triton-X-100, 1 mM PMSF#

High-salt buffer: 50 mM HEPES, pH 7.5, 1M NaCl, 10 mM MgCl2, 100 U/mlDNAse I

Sarcosyl buffer: 50 mM HEPES, pH 7.5, 0.5% sarcosyl

Other Buffers:

-   A. 50 mM HEPES pH 7.5, 300 mM NaCl, 250 mM sucrose, 5 mM EDTA, 5 mM    GSH, 1% NP-40, 0.2% sarcosyl.-   B. 50 mM HEPES pH 7.5, 1.5 M NaCl, 250 mM sucrose, 5 mM EDTA, 5 mM    GSH, 1% NP-40.-   C. 50 mM Tris pH 8, 250 mM sucrose, 5 mM MgCl₂, 5 mM GSH, 1% NP-40.-   D. 50 mM HEPES pH 7.5, 5 mM EDTA, 5 mM GSH, 1% NP-40.-   E. 50 mM HEPES pH 7.5, 2.3 M sucrose, 5 mM EDTA, 5 mM GSH, 1% NP-40.    Adjust sucrose concentration using Puffer D.-   F. 50 mM HEPES pH 7.5, 150 mM NaCl, 250 mM sucrose, 5 mM EDTA, 5 mM    GSH, 2×Pls.

Abbreviations: Pis=protease inhibitor cocktail (Roche);PMSF=phenyl-methylsulfonyl fluoride (Sigma), GSH=reduced glutathione(Sigma)

Protocol 1: “Preparation of the insoluble protein fraction (misfoldedproteome) from a 10% brain homogenate”

1. Shock-frozen brain fractions from brain regions BA8, BA9, and BA23,BA24 of deceased patients, who had been diagnosed with schizophreniawhen they were alive, were used. BA8, BA9, BA23, and BA24 refer toso-called Brodmann areas and correspond to certain neuronal centers inthe neocortex.2. Weigh out brain samples on dry ice and homogenize in thecorresponding volume of VRL buffer. Store at −80° C.3. Thaw the homogenate on ice and centrifuge in the presence of 0.5%Triton-X-100 in 2 ml micro-reaction vessels at 20,000 g at 4° C. for 20min. Collect supernatant and resuspend pellet in the same volume of VRLbuffer plus Triton.4. Repeat the centrifugation as above. Add supernatant to firstsupernatant, store 500 μl separately at −80° C.5. Dissolve pellets in a total of 4 ml high sucrose buffer. Subject toultracentrifugation in ultra-clear centrifuge tubes 5 ml at 130,000 g,4° C., 45 min (Beckmann MLS-50, 40,000 rpm), collect supernatant, freezesupernatant lipid layer in separate microreaction vessel.6. Resuspend pellet in another 4 ml of high-sucrose buffer and repeatcentrifugation as above. Add supernatant to the first supernatant.Storage at −20° C.7. Dissolve pellet in 4 ml high-salt buffer, incubate over-night at 4°C. Subject to ultracentrifugation in ultra-clear centrifuge tubes 5 mlat 130,000 g, 4° C., 45 min (Beckmann MLS-50, 40,000 rpm), collectsupernatant, and resuspend pellet again in high-salt buffer (withoutDNAse). Upon need, take up pellet with an insulin syringe equipped witha 0.6 mm to 0.4 mm-cannula.8. Second centrifugation as above. Add supernatant to first supernatant,store 500 μl separately at −80° C. Storage at −20° C.9. Dissolve pellet in 200 μl sarcosyl buffer. For this purpose, mincepellet in 100 μl buffer using the pipette tip and then transfer to a 0.5ml microreaction vessel. Rinse vessel and pipette tip with another 100μl sarcosyl buffer and add to first 100 μl. Use insulin syringe and 0.4mm-cannula to dissolve pellet. Incubate in rotator at 4° C. for approx.1 h. Possibly, after approx. half of this time, rehomogenize using aninsulin syringe.10. Ultracentrifugation in microreaction vessel (Beckmann 357448polyallomer tubes with snap-on caps) whose weight was determined on ananalytical scale, at 112,000 g, 4° C., 45 min) (Beckmann TLA-55, 50,000rpm). Collect supernatant and wash again in 200 μl sarcosyl buffer.Resuspension and centrifugation as above.11. Collect supernatant and determine the weights of the pellets.Storage at −80° C.

Protocol 2“Preparation of the insoluble protein fraction (misfoldedproteome) from a 10% brain homogenate”

1. Homogenize the brain fragment (e.g. 0.3-0.4 gr=1 vol) at 5% (w/v) inbuffer A (plus 2×Pls, 1 mM PMSF) and centrifuge at 1,800×g, 30 min, 4°C. Wash the pellet in buffer A (5 ml [15 vol] washing volume) andcontinue the work-up.

2. Suspend the pellet in buffer B (10 ml [30 vol] washing volume; andadd 1 mM PMSF and 0.2% sarcosyl), centrifuge (1,800×g, 30 min, 4° C.),and wash once in buffer B (5 ml [15 vol] washing volume).

3. Continue the work-up of the pellet after washing in buffer C (5 ml[15 vol] washing volume), then resuspend thoroughly in buffer C (5 ml[15 vol] washing volume; plus 2×Pls, 1 mM PMSF, benzonase, and DNAsel,40 U/ml each) and shake for 30 minutes at 37° C. Subsequently, shake thesame mixture over-night at 4° C. and centrifuge the next morning(1,800×g, 30 min, 4° C.).4. Subsequently, wash the pellet in buffer A (plus 1 mM PMSF, (5 ml [15vol] washing volume), centrifuge (1,800×g, 30 min, 4° C.), and resuspendin buffer D. After successful resuspension, add sucrose to adjust thefinal sucrose concentration to 1.6 M (70% of the sucrose concentrationof buffer E).5. The suspension obtained under 4) is then placed at a ratio of 4:1 ona bed of 1 ml of buffer E and centrifuged in the ultracentrifuge at45,000 rpm for 45 minutes using the MLS-50 rotor (approx. 120,000×g).6. Remove the interphase between the sucrose phases with a pipette(approx. 1 ml) and dilute with buffer D at a ratio of 1:4. Then placethis suspension again on 1 ml 70% buffer E and centrifuge for 45 minutesat 45,000 rpm in the MLS-50 rotor (approx. 120,000×g). Take up theresulting pellet (0.1 ml) in buffer F.7. The resulting pellet (“insoluble protein fraction”) is subsequentlyused both for immunization and for the dot blots and SDS-PAGE/Westernblot.

All steps are carried out at 4° C., and/or on ice. The centrifugation insteps 1-4 is carried out with a tabletop centrifuge at 1,800×g, 30minutes at 4 degrees centigrade.

B. Antibody Production

a) Polyclonal Antibodies

Chickens, rabbits, and mice (BALB/c) are immunized with approx. 500-1000μg/100 μl of pellets pooled from four schizophrenia patients. In thiscontext, RIBI (Sigma) is added to the (aggregated) antigen as adjuvant.The animals are boostered twice with an interval of 3 weeks. The immuneresponse is investigated two weeks after the final booster. In the caseof chickens, eggs are collected one week after the booster, startingafter the first booster, and antibodies (IgY) are isolated from the eggyolk using standard methods.b) Monoclonal AntibodiesIn order to obtain monoclonal antibodies, a suitable animal is immunizedas described and antibody-producing cells (e.g. B cells from the spleen)are removed from the immunized animal in known fashion (G Köhier, CMilstein 1975, Continuous cultures of fused cells secreting antibody ofpredefined specificity. Nature, 256, 495-497), fused with immortalizedmyeloma cells, and subjected to selection. The hybridoma cells obtainedare then selected with regard to the specificity for the misfoldedprotein of the antibodies they produce.

The monoclonal antibodies 7B2 and 9C9 were produced as follows:Insoluble misfolded proteins purified from frozen pieces of brain(cortex, BA8) of 15 schizophrenia patients were pooled and injectedsubcutaneously into prion protein (PrP) knockout mice for immunizationusing RIBI as adjuvant. PrP knockout mice were used since they have beenused successfully for generating conformation-specific monoclonalantibodies before, and they are also used preferably in the presentinvention for generating conformation-specific mABs against antigensother than PrP. The mice were boostered twice, after an interval ofthree weeks each; ten days after the final booster, the mice received anintraperitoneal booster on two consecutive days, and the spleen wasremoved for fusion on the third day. The spleen cells (splenocytes) werefused with the myeloma cells according to standard methods to obtainresulting hybridoma cells.

C: Immunological Characterization

In general, the antibodies obtained were used to investigate brainhomogenates of normal, schizophrenic, depressive, and bipolar affectivedisorder patients by means of Western blotting and/or dot blotting.

The results are shown in the figures.

In the figures:

FIG. 1 shows a Western blot of biochemically fractionated pellet orfirst supernatant (after sarcosyl incubation) of brain homogenates ofnormal or schizophrenic patients after biochemical fractionation forpoorly soluble, sarcosyl-resistant protein aggregates (antibody: chickenIgY).

S=first supernatant, P=pellet, N1-N4=brain of healthy subjects,S1-S4=brain of schizophrenia patients. The arrows (p85ch, p58ch, p20ch)refer to immunoreactive bands that occur specifically in schizophreniapatients only and, thus, constitute biological markers. The arrows onthe left side indicate molecular weights.

FIG. 2 shows a Western blot of biochemically fractionated pellet orfirst supernatant of brain homogenates of normal or schizophrenicpatients after biochemical fractionation for poorly soluble,sarcosyl-resistant protein aggregates (antibody: mouse serum).

S=first supernatant, P=pellet, N1-N4=brain of healthy subjects,S1-S4=brain of schizophrenia patients. The arrows refer toimmunoreactive bands that occur specifically in schizophrenia patientsonly and, thus, constitute biological markers thereof (p55mo, p35mo).The arrows on the left side indicate molecular weights.

FIG. 3 a shows a dot blot using monoclonal antibody RC1 undernon-denaturing conditions of a pool of sarcosyl-resistant pellet(fraction X) of pooled normal brain (N; BA9) and pooled schizophreniabrain (S; BA9).

The monoclonal conformation-specific antibody, RC1, that recognizes withhigh conformational specificity the native surface structure of aprotein that is specifically present in schizophrenia, was used asantibody.

FIG. 3 b shows a dot blot assay with monoclonal antibodies 7B2 and 9C9.The monoclonal antibodies, 7B2 and 9C9, were obtained and testedaccording to the following screening: the cell culture supernatants ofapprox. 3,000 hybridoma cells were screened using a dot blot assay and aspecialized apparatus (ELIFA apparatus; Pierce, USA) with a 96-wellformat. In the process, identical quantities of the pooled insolubleproteins (purified according to protocol 1) from schizophrenia patientsand normal people were applied to nitrocellulose in parallel, themembrane was blocked with 5% non-fat dry milk in TBST (Tris-bufferedsalt solutions containing Tween), and the cell culture supernatants inthe wells of the ELIFA apparatus were incubated on the dots at roomtemperature for 2 h. The membrane was washed and incubated for one hourwith a secondary antibody (anti-mouse IgG/M) to which peroxidase wascovalently coupled. The blot was then washed, ECL substrate was added,and the blot was developed on hyperfilm (Amersham). The results areshown in FIG. 3 b. The hybridoma cells of the supernatants that reactedmuch more strongly or exclusively with the insoluble proteins of theschizophrenia brains (black) were picked and sub-cloned multiply. Then,a substantial quantity of supernatant in serum-free medium (PFHM; Gibco,USA) was produced and used in the subsequent tests. Two antibodiestermed 7B2 and 9C9 proved to be particularly well suited.

FIG. 4 shows a Western blot of biochemically fractionated pellet ofbrain homogenates of normal or schizophrenic patients.

The homogenates were purified according to protocol 1. Antibodies werefrom mouse serum. P=pellet, N1-N4=brain of healthy subjects, S1-S4=brainof schizophrenia patients. The arrows indicate immunoreactive bands thatoccur specifically in schizophrenia patients only and thus constitutebiological markers (p45mo2, p37mo2). Antibodies: mouse serum.

FIG. 5 a) shows a Western blot of biochemically fractionated pellet ofbrain homogenates of normal or schizophrenic patients.

The homogenates were purified according to protocol 1. The monoclonalantibody, AK SX16.3, was used as antibody. P=pellet, N1-N4=brain ofhealthy subjects, S1-S4=brain of schizophrenia patients. The arrowsindicate immunoreactive bands that occur specifically in schizophreniapatients only and thus constitute biological markers (p37, p-stack).p-stack is an immunoreactivity from the well. This corresponds toinsoluble proteins that had been taken up when the gel was loaded, butwere not transported in the gel due to their insolubility. Some ofp-stack dissolved and forms p37.

FIG. 5 b) shows a Western blot of biochemically fractionated pellet ofbrain homogenates of normal or schizophrenic patients from the region,BA9.

The homogenates were purified according to protocol 1. The monoclonalantibody, AK 7B2, was used as antibody. P=pellet, N1-N4=brain of healthysubjects, S1-S4=brain of schizophrenia patients. It is evident that AK7B2 shows immunoreactivity only in the case of schizophrenia brains.

FIG. 6 shows a Western blot of biochemically fractionated pellet and/orthe first supernatant of brain homogenates of normal or schizophrenicpatients.

The homogenates were purified according to protocol 1. Antibodies werefrom rabbit antiserum. S=first supernatant, P=pellet, N1-N4=brain ofhealthy subjects, S1-S4=brain of schizophrenia patients. Although thereare no immunoreactive bands that occur exclusively in schizophrenics,but not in healthy people, it is clearly evident that, in the markedrectangles, immunoreactivity is present only in schizophrenics. Thismeans that insoluble proteins pellet in the molecular region below 60 kDexclusively in schizophrenics.

FIG. 7 shows a Western blot of biochemically fractionated pellet and/orthe first supernatant of brain homogenates of normal or schizophrenicpatients.

The homogenates were purified according to protocol 1. The monoclonalantibody, MW1, recognizing polyglutamine-containing (“polyQ”) epitopes[Ko et al., 2001, Brain Research Bulletin 56:319f] was used as antibody.S=first supernatant, P=pellet, N1-N4=brain of healthy subjects,S1-S4=brain of schizophrenia patients. BA9 and BA24 denote various brainregions according to Brodman. Polyglutamine-containing bands are evidentat the upper edge of the gel and correspond to SDS-resistantpolyglutamine multimers pelleted in the well that had been taken upduring the loading of the gel, but were not transported in the gel dueto their relative insolubility. However, no difference between normaland schizophrenia patients can be detected. Therefore, this is evidenceto indicate that polyglutamine-containing proteins are notschizophrenia-specific.

FIG. 8 shows a Western blot using 7B2 and 9C9 against insoluble proteinfractions B8 of the SMRI Consortium Collection (SMRI).

The monoclonal antibodies, 7B2 and 9C9, were tested on the Western blot(WB) against the insoluble protein fractions of BA8 brain homogenates ofpatients afflicted by schizophrenia (the same that were used forimmunization), depression, bipolar disorder, and normal controls. Theresults are shown in FIG. 8. The insoluble protein fraction of theindividual brains was separated by means of SDS-PAGE and shown in theWB. The assessment was made blinded, i.e. without the above-mentioneddiagnoses of the individual brains being known. A positive assessmentwas made only in the case of bands possessing sufficientimmunoreactivity. With regard to mAB 9C9, statistical analysis by meansof crosstabs/Pearson chi-square analysis showed that the immunoreactivebands assessed as positive recognized the following: 2 (of 15) normalcontrols; 5 (of 15) depressive patients, 7 (of 15) bipolar patients, and7 (of 15) schizophrenics. Thus, 9C9 afforded significant recognition ofdiseased vs. normal (p=0.042), schizophrenic vs. normal (p=0.046), andbipolar vs. normal (p=0.046). SPSS (version 11.0 on Apple G4) was usedfor statistical analysis.

With regard to mAB 7B2, statistical analysis by means ofcrosstabs/Pearson chi-square analysis showed that the immunoreactivebands assessed as positive recognized the following: 2 (of 15) normalcontrols; 6 (of 15) depressive patients, 5 (of 15) bipolar patients, and7 (of 15) schizophrenics. Thus, 7B2 afforded significant recognition ofschizophrenic vs. normal (p=0.046), but not of bipolar vs. normal(p=0.195) or diseased vs. normal (p=0.058). SPSS (version 11.0 on AppleG4) was used for statistical analysis.

FIG. 9 shows a Western blot of 7B2 against insoluble protein fractionsfrom BA23 (SMRI)

Moreover, 7B2 was tested on the Western blot (image 3) against theinsoluble protein fractions of BA23 brain homogenates, i.e. from adifferent brain region, of patients afflicted by schizophrenia,depression, bipolar disorder, and normal controls. A positive assessmentwas made only in the case of bands that possessed sufficientimmunoreactivity and, in particular, included the second, somewhatsmaller, band.

With regard to mAB 7B2, statistical analysis by means of crosstabsPearson chi-square analysis showed that the immunoreactive bandsassessed as positive recognized the following: 4 (of 15) normalcontrols; 9 (of 15) depressive patients, 10 (of 15) bipolar patients, 4(of 15) schizophrenics. Thus, 7B2 afforded significant recognition ofbipolar vs. normal (p=0.028), but not of schizophrenia vs. normal (p=1)or diseased vs. normal (p=0.09). SPSS (version 11.0 on Apple G4) wasused for statistical analysis.

Comparison of the data from FIG. 8 and FIG. 9 therefore shows that the7B2 antigen is insoluble in the BA8 region (prefrontal cortex) andallows a distinction to be made between schizophrenia brains and normalbrains in this region, but that this does not apply in another brainregion (BA23, posterior cingulum). In this site, 7B2 cannot distinguishbetween schizophrenia brains and normal brains, but between bipolarbrains and normal brains, which it does not afford in the BA8 region.

These results reflect the fact that there are overlaps in the biologicalcause of schizophrenia and bipolar disorder that are reflected in thedifferent solubility of the antigen of 7B2 in various brain regions.These differences might be caused by the differential presence ofcertain cell types or a certain extracellular milieu. In summary, it canbe concluded that mABs 7B2 and 9C9 are suitable for specific recognitionof neuropsychiatric diseases, specifically of schizophrenia, bipolardisorder, and depression, by means of brain homogenates.

Deposition of Biological Material:

Hybridoma cells producing the antibodies, 7B2 and 9C9, have beendeposited with “Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, Mascheroder Weg 1 b, D38124 Braunschweig”, in compliance with theBudapest agreement as follows:

1. Hybridoma cells (antibody 7B2): DSM ACC2713, deposition date:26.01.2005.

2. Hybridoma cells (antibody 9C9): DSM ACC2714, deposition date:26.01.2005.

1. A monoclonal antibody produced from the hybridoma cell line depositedas DSM ACC2714 (mAB 9C9).
 2. The monoclonal antibody according to claim1, wherein the monoclonal antibody recognizes misfolded proteins thatare associated with schizophrenia.
 3. The monoclonal antibody accordingto claim 1, wherein the monoclonal antibody recognizes misfoldedproteins that are associated with depression.
 4. The monoclonal antibodyaccording to claim 1, wherein the monoclonal antibody recognizesmisfolded proteins that are associated with bipolar affective disorder.5. The monoclonal antibody according to claim 1, wherein the monoclonalantibody has been modified to be a blood-brain barrier-crossingmonoclonal antibody.
 6. A binding fragment of a monoclonal antibodyproduced from the hybridoma cell line deposited as DSM ACC2714 (mAB9C9), wherein said binding fragment recognizes misfolded proteins thatare associated with neuropsychiatric diseases selected from the groupconsisting of schizophrenia, depression and bipolar affective disorder.7. The monoclonal antibody according to claim 1, wherein the monoclonalantibody is coupled to a pharmaceutically active substance.
 8. Themonoclonal antibody according to claim 1 wherein the monoclonal antibodyis coupled to an isotope or a radioactive labeled molecule.
 9. Apharmaceutical preparation in a blood-brain barrier-crossing formcomprising the monoclonal antibody according to claim
 1. 10. Thepharmaceutical preparation according to claim 9, wherein the monoclonalantibody is coupled to a pharmaceutically active substance.
 11. Thepharmaceutical preparation according to claim 9, wherein the monoclonalantibody is coupled to an isotope or a radioactively labeled molecule.12. The binding fragment according to claim 6, wherein the bindingfragment is cloned into a chimeric antibody or is combined withhumanized or human sequences.
 13. The binding fragment according toclaim 6, wherein the binding fragment is coupled to a pharmaceuticallyactive substance.
 14. The binding fragment according to claim 6, whereinthe binding fragment is coupled to an isotope or a radioactive labeledmolecule.
 15. A method for diagnosing a neuropsychiatric diseaseselected from the group consisting of schizophrenia, depression andbipolar affective disorder using a monoclonal antibody, or bindingfragment thereof, produced from the hybridoma cell line deposited as DSMACC2714 (mAB 9C9) that recognizes misfolded proteins that are associatedwith said neuropsychiatric disease, said method comprising: a)contacting a tissue or body fluid sample of a patient with saidmonoclonal antibody or binding fragment thereof; and b) detecting anyantibody-protein complexes thus formed, wherein the presence of saidantibody-protein complexes is considered to be a positive finding forsaid neuropsychiatric disease.
 16. The method according to claim 15,wherein the presence of antibody-protein complexes is detected by meansof ELISA, Western blotting or immuno-coupled fluorescence methods.